Assay for p53 function in cells

• US 20030228627A1
• Filed: 03/24/2003
• Published: 12/11/2003
• Est. Priority Date: 03/22/2002
• Status: Abandoned Application

• Current Assignee: Salk Institute for Biological Studies
• Original Assignee: Salk Institute for Biological Studies
• Inventors: Espinosa, Joaquin, Emerson, Beverly M.
• Application Number:
  US10/397,131
• Publication Number:
  US 20030228627A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 2510/00 Detection of programmed cell death, i.e. apoptosis
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

Disclosed herein is an assay for identifying compounds that modulate the interaction between p53 and specific DNA binding sites. The assay uses chromatin-assembled target genes to correctly reproduce p53 binding to target genes of interest. This assay permits the selection of compounds which preferentially associate with p53 when bound to specific DNA binding sites but not others. Moreover, compounds can be screened for their ability to selectively modulate p53 interaction with specific binding sites associated with individual genes. Compounds can also be screened for their ability to selectively modulate p53 interaction with specific cofactors (proteins, nucleic acids, peptides, etc.) when p53 is bound to specific DNA sequences. This system allows drugs to be tailored to affect p53-dependent regulation of certain target genes that regulate distinct cellular pathways without affecting other genes and pathways.

Claims

1. A method for identifying compounds that modulate the binding of p53 protein to a gene of interest, comprising:

providing said gene of interest as chromatin-assembled DNA;

contacting said gene of interest with said p53 protein and a test compound; and

determining whether the presence of said test compound modulates the binding of said p53 to said gene of interest.

2. The method of claim 1, wherein said p53 protein comprises a human p53 protein.

3. The method of claim 1, wherein said p53 protein comprises recombinant p53 protein.

4. The method of claim 1, wherein said p53 protein comprises p53 protein that is purified from tissues.

5. The method of claim 1, wherein contacting said gene of interest further comprises contacting said gene of interest with a cell nuclear extract.

6. The method of claim 1, wherein said gene of interest is a cell cycle control gene or an apoptotic gene.

7. The method of claim 1, wherein said p53 protein is a full-length p53 protein.

8. The method of claim 1, further comprising determining whether the presence of said test compound modulates p53 controlled transcription of said gene of interest.

9. The method of claim 1, wherein determining whether the presence of the test compound modulates the binding of p53 comprises a footprinting assay, an electrophoretic mobility shift assay or an chromoimmunoprecipitation assay.

10. The method of claim 1, wherein said test compound is selected from the group consisting of:

proteins, peptides, antibodies, small organic molecules, and inorganic molecules.

11. The method of claim 1, wherein determining whether the presence of the test compound modulates the binding of p53 comprises determining whether the presence of the test compound decreases the binding of p53.

12. The method of claim 1, wherein determining whether the presence of the test compound modulates the binding of p53 comprises determining whether the presence of the test compound increases the binding of p53.

13. A method for identifying a modulator that inhibits growth of cancer cells, comprising:

providing a gene construct comprising a chromatin-assembled promoter linked to a reporter gene, wherein said promoter binds p53 protein;

incubating said gene construct in the presence of p53 protein and a test compound; and

determining whether said test compound increases binding of said p53 protein to said gene construct by measuring the amount of reporter gene activity in the presence and the absence of said test compound.

14. The method of claim 13, wherein said promoter comprises the p21 promoter.

15. The method of claim 13, wherein said reporter gene comprises the luciferase gene.

16. The method of claim 13, wherein said modulator is selected from the group consisting of:

proteins, peptides, antibodies, small organic molecules, and inorganic molecules.

17. The method of claim 13, wherein said cancer cells are tumor cells.

18. The method of claim 13, wherein said promoter is a pro-apoptotic promoter.

19. A method for identifying a test compound that modulates the interaction of p53 protein with a transcription cofactor that associates with p53 protein when p53 protein is bound to a gene of interest, comprising:

providing said gene of interest as chromatin-assembled DNA;

contacting said gene of interest with said p53 protein, the transcription cofactor, and the test compound; and

determining whether the presence of said test compound modulates the association of said cofactor with said p53 protein bound to said gene.

20. The method of claim 19, wherein said cofactor is purified from an expression system.

21. The method of claim 19, wherein said cofactor is present in a tissue extract.

22. The method of claim 21, wherein said tissue extract is a protein extract.

23. The method of claim 21, wherein said extract is from a tissue affected by a neoplastic, autoimmune, or neurodegenerative disorder.

24. The method of claim 21, wherein said tissue extract is from a cell treated with a DNA damaging agent.

25. The method of claim 19, wherein said gene of interest comprises a pro-apoptotic promoter, a cell cycle promoter or a tissue specific promoter.

26. The method of claim 19, wherein said gene of interest comprises a p21 promoter.

27. The method of claim 19, wherein said transcriptional cofactor is selected from the group consisting of:

histone acetyl transferase cofactor p300, Pit-1, MDM2, TAFs, TRAP, the CREB binding protein (CBP) and p300 (CBP/p300), and both CBP/p300 and p300/CBP-associated factor (PCAF).

28. A kit for identifying test compounds that affect p53 protein binding to a promoter, comprising:

a gene construct comprising a chromatin assembled p53 binding site genetically linked to a reporter gene; and

isolated p53 protein.

29. The kit of claim 28, wherein said reporter gene encodes a detectable protein.

30. The kit of claim 29, wherein said detectable protein is an enzyme or a fluorescent protein.

31. The kit of claim 30, wherein said enzyme is luciferase.

32. The kit of claim 30, wherein said fluorescent protein is a green fluorescent protein.

33. The kit of claim 28, further comprising at least one transcriptional cofactor.

34. The kit of claim 33, wherein said transcriptional cofactor is selected from the group consisting of:

histone acetyl transferase cofactor p300, Pit-1, MDM2, TAFs, TRAP, the CREB binding protein (CBP) and p300 (CBP/p300), and both CBP/p300 and p300/CBP-associated factor (PCAF).

35. The kit of claim 33, wherein said transcriptional cofactor is isolated from a nuclear extract.